Lung cancer is a prevalent disease and consumes many lives every year. Disease relapse, invasion and metastases are the main causes of death. Recent discoveries provide compelling evidence that at least some types of cancer are initiated and maintained by a small population of malignant cells called cancer-initiating stem cells (CICs). Relapse, invasion, and metastases are explained by the fact that CICs have a different biology than all the other tumor cells and, importantly, are resistant to chemotherapies and radiation. Lung CICs have been shown to represent about 1-15% of all tumor cells and can form tumors with injections as low as 100 cells. Evidence from published studies have demonstrated that human, as well as rodent, cancers contain populations of cells that express embryonic stem (ES) cell antigens. Cells containing these proteins also express markers used to identify lung CICs; therefore, we hypothesized that ES cells and CICs share several common molecular traits. To test this hypothesis, we vaccinated mice with irradiated, allogeneic murine ES cells in combination with a source of granulocyte-macrophage colony stimulating factor (GM-CSF) as an immunostimulatory adjuvant (ES cell vaccine) and investigated whether an anti-tumor immune response was elicited. We discovered that ES cell vaccination is very effective in preventing both implantable and carcinogen-induced lung adenocarcinoma development without any detectable toxicity or signs of autoimmunity. Preliminary studies from our laboratory reveal that splenocytes from ES cell-immunized mice are preferentially cytotoxic to lung CICs. Experiments proposed in this application seek to expand these novel findings to convincingly demonstrate that ES cells immunize against lung cancer- associated CICs and that anti-CIC immunity is responsible for preventing lung adenocarcinoma development. A major goal of this study is to assess the potential of CIC-targeting ES cell vaccine as a treatment option for lung cancer patients. We will test the translational potential of our approach using a novel ES cell-derived exosome- based vaccination strategy (Es-exo vaccine). Experiments proposed in this study will address the in vivo efficacy of ES-exo vaccine as a cell-free vaccine modality in both prophylactic and therapeutic settings against lung cancer. The biological features responsible for the anti-tumor activities of ES-exo vaccine as well as their immunostimulatory properties will be investigated in in vitro and in vivo lung cancer mouse models. To fulfill the stated objectives, the following aims are proposed: 1) Investigate whether lung cancer-initiating cells are targets of ES cell vaccination- induced anti-tumor immunity. 2) Evaluate the translational potential of ES cell-derived exosomes (ES-exo) as a novel cell-free vaccine for lung cancer. Our proposed study will provide important insights towards developing a safe immunotherapeutic vaccine for lung cancer onset and/or recurrence.